1. Field of the Invention
The present invention relates to the enzyme solanidine UDP-glucose glucosyltransferase (SGT) which is involved in the biosynthesis of steroidal glycoalkaloids in solanaceous plants. More particularly, the invention relates to DNA sequences which encode SGT, recombinant polynucleotide molecules containing the sequences, and use thereof, in particular, use of an antisense DNA construct to inhibit SGT activity and glycoalkaloid levels in solanaceous plants.
2. Description of the Art
Solanaceous plants include such agronomically important crops as potato and tomato. Solanaceous species synthesize steroidal glycoalkaloids (nitrogen-containing steroidal glycosides) which are natural toxicants and are believed to serve as natural defenses against insect and other pests. These compounds can exhibit toxic effects in humans as well as insects, and thus represent a potential source of toxicants, especially in improperly stored or processed potatoes. This has led to the implementation of a guideline limiting glycoalkaloid content in a tuber of a given potato cultivar to 20 mg/100 gm. Worldwide, between 13 and 27% of the potato crop has to be discarded because glycoalkaloid levels are above the maximum currently deemed to be safe (Morris and Lee, Food Technol. Aust. 36: 118-124 (1984)). While the guideline provides effective protection for the consumer, its effectiveness is dependent upon limiting the release of new cultivars for commercial production to those with acceptable glycoalkaloid levels. For potato breeding programs to develop new cultivars with improved agronomic or processing properties, the need to select also for low levels of glycoalkaloids can present a difficult problem. A method to decrease the glycoalkaloid content of any newly developed cultivar with minimum impact on other characteristics would be of great use to obtain valuable new commercial potato cultivars.
In cultivated potato the predominant glycoalkaloid species, .alpha.-chaconine and .alpha.-solanine, are triglycosylated derivatives of the aglycon solanidine. These steroidal glycoalkaloids (SGAs) contain either glucose (.alpha.-chaconine) or galactose (.alpha.-solanine) as the primary glycosyl residue. A simplified biochemical pathway illustrating biosynthesis of the toxic glycoalkaloids .alpha.-chaconine and .alpha.-solanine is shown in FIG. 1. The synthesis of .gamma.-chaconine (3-.beta.-O-glucosylsolanidine), an intermediate in the pathway to .alpha.-chaconine, is catalyzed by SGT. The activity and kinetics of the enzyme SGT have been characterized by a number of groups (Stapleton et al., J. Agric. Food Chem. 39:1187-1193 (1991); Bergenstrahle et al., Plant Sci. 84:35-44 (1992); Zimowski, Phytochemistry 6:1827-1831 (1991); Packowski and Wojciechowski, Phytochemistry 35:1429-1434 (1994)). The aglycone portion of the glycoalkaloid is believed to be considerably less toxic than the glycoside. It is believed that decreasing the activity of the enzyme(s) responsible for glycosylation of the aglycone should effectively lower the potential toxicity of potato cultivars.
A biosynthetic pathway to solanidine has been proposed (Kaneko et al., Phytochemistry 15: 1391-1393 (1976), E. Heftmann, Phytochemistry 22:
1843-1860 (1983)). Information on the enzymatic mechanisms involved in the glycosylation steps of solanidine to form glycoalkaloids is limited to reports utilizing relatively crude enzyme preparations (D. R. Liljegren, Phytochemistry 10: 3061-3064 (1971), Jadhav et al., Journal of Food Science 38: 1099 (1973), Lavintman et al., Plant Science Letters 8: 65-70 (1977), Osman et al., Phytochemistry 19: 2599-2601 (1980), J. Zimowski, Phytochemistry 30: 1827-1831 (1991)). Stapleton et al., 1991, supra, reported a 600-fold purification of a monomeric, 36- to 38-kilodalton (kDa), soluble protein, SGT from potato sprouts. SGT was isolated by anion-exchange ("Mono Q"), size exclusion ("Superose" 12), and chromatofocusing ("Mono P"). This purification protocol resulted in a very low yield of SGT. A major difficulty encountered was the copurification of SGT with patatin. Patatin is an approximately 40-kDa glycoprotein which can constitute up to 40% of the soluble potato tuber protein.